The term “microorganism” covers any microorganism that could react to its exposure to an antibiotic, such as bacteria or yeasts. Although the invention is more specifically described hereinafter with reference to bacteria, it is meant that it is not restricted thereto.
This determination is of major interest in the microbiological diagnosis in the fields of health, agri-food, environment, it can be just as important in pharmacology, in the screening of new molecules, in particular antibiotics, or in the search for cytotoxic compounds present in food products, for example milk. This selection of applications is not exhaustive, and in general, the invention can find application in any field the moment the question was raised of the reaction of cells to an exposure to a chemical or biological compound.
The Raman effect is a light diffusion phenomenon that applies to the vast majority of molecules. Its observation in spectroscopy allows characterizing a molecule, a microorganism, a medium, it is of a simple implementation, it is fast and cost-effective, and has the substantial advantage in biology of not being strongly disrupted by water and of not requiring a labelling or a contrast agent.
Thereby, according to the document AIM Athamneh et al. (2014) Antimicrob Agents Chemother 58:1302-1314, the authors used Raman spectroscopy in order to characterize the sensitivity of E. coli cultures to 15 known antibiotics representing 5 families of antibiotics, in order to constitute a baseline. For this purpose, the described method comprises the following steps, producing an E. coli culture, exposing a sample of this culture to antibiotics in a concentration of triple the minimum inhibitory concentration (MIC), maintaining said cells in contact with the antibiotic for at least 30 minutes, harvesting and washing bacterial cells, collecting a cell suspension and treating for analyzing the cell layers by Raman spectroscopy. The result of the analysis is derived from the average of a multiplicity of spectra obtained for each collection comprising a multitude of cells and integrated into the reference base. Once constituted, this reference base can be exploited in order to allocate an unknown antibiotic to one of said 5 families, depending on the sensitivity of an E. coli culture to that antibiotic. The classification performances obtained accordingly allow obtaining elements as to the class of the unknown molecule, which can be useful for pharmacological research. The obtained results, however, do not provide information related to the sensitivity phenotype of the studied bacteria or information that is conducive to clinical use.
The document WO2013/093913A1 describes a method for identifying a bacterium in a biological fluid using in particular Raman spectroscopy. A sample of a bacterial culture of said biological fluid is submitted to an incident light and the resultant light obtained by diffusion is analyzed by Raman spectroscopy. The read signal is then interpreted thanks to a reference base listing the spectral signature of different microorganisms defined in the same conditions. This method may further comprise a step of exposing said bacteria to an antibiotic, the read signal being an effect of said antibiotic on those bacteria. The measured effect of the antibiotic is exerted in particular on the viability of the bacterial cells or on the development of the culture. The disadvantage of this method lies in its use of a culture step which not only protracts the acquisition of an answer, but also, moreover, requires mastering an additional step necessary for obtaining the expected answer.
If using Raman spectroscopy allows lightening the determination of the sensitivity clinical profile of bacteria of interest to an antibiotic, the fact remains that, according to this prior art, it is applied to a bacterial culture whose obtaining within a time of about 18 to 24 hours, does not allow accessing a rapid determination method. In diagnosis, this constitutes a major obstacle to an effective care of patients.
According to the document U. Münchberg et al. (2014) Anal Bioanal Chem 406: 3041-3050, the authors raise the problem of the rapid establishment of an appropriate antibiotic treatment in a patient, as well as the difficulties encountered by techniques using a cell culture when the patient has already received an antibiotic treatment. The authors then dispense with the culture step and apply Raman spectroscopy to individual bacteria. This work therefore addresses the issue of identifying bacteria previously exposed to an antibiotic, a potential source of misdiagnosis. In order to solve this problem, the authors constitute a reference base comprising the results of Raman analysis performed on individual cells which were not exposed to an antibiotic and on cells which were exposed to an antibiotic, in various concentrations lower than the minimal inhibitory concentration (MIC). The conclusion of this study is an absence of major difficulty in identifying the bacteria under these conditions. The authors did not notice any significant effect of the antibiotics on the bacterial spectra and conclude that the possible modifications are observed in areas of high variability and therefore unusable.
None of these solutions allows considering a reliable method for determining the sensitivity clinical profile of a bacterium of interest to an antibiotic and in particular determining its resistance or sensitivity to an antibiotic, in a short time in the range of a few hours which would allow having a diagnosis within the day. This lack is responsible for ineffective antibiotic therapy, for risk of worsening the infection of the patient and for a difficulty in establishing an accurate diagnosis when the patient has already been treated. This lack is all the more felt at the time of emergence and spread of multi-antibiotic resistant bacteria.